Question

If we had used a buffer of pH11 in our IEX experiment, we would have observed no binding between myoglobin and the DEAE-beads. Even though myoglobin should hold a negative net charge at pH11, explain why there would be no binding

Answer 1

At higher pH myoglobin should bind the DEAEW beads but it is not happening in this case. THe probable reasons are:

The most appropriate reason is that if a buffer has much higher pH than the pI of protein then it will remain unionised and will not elute. SO the pH of the buffer should be near to or slightly higher than the protein for proper elution.

High salt concentration may be present in the loading buffer as the pH is too high. This salt concentration may inhibit the binding of myoglobin to the DEAE beads.

the pI is not attained properly. Actual pI of the protein is different from the theoretical value. So, it need to moniter the solubility of the protein and Isoelectric focalisation is the best method to calculate the actual pI.

And the other possible reason is that DEAE beads can bind a protein maximum upto a pH of 8. pH 11 is very high whch may n